Overcoming Species Barriers: an Outbreak of Lagovirus Europaeus GI.2/RHDV2 in an Isolated Population of Mountain Hares (Lepus Timidus) Aleksija S

Neimanis et al. BMC Veterinary Research (2018) 14:367 https://doi.org/10.1186/s12917-018-1694-7

RESEARCH ARTICLE Open Access Overcoming species barriers: an outbreak of Lagovirus europaeus GI.2/RHDV2 in an isolated population of mountain hares (Lepus timidus) Aleksija S. Neimanis1,2* , Harri Ahola3, Ulrika Larsson Pettersson1, Ana M. Lopes4,5, Joana Abrantes4, Siamak Zohari3, Pedro J. Esteves4,6,7 and Dolores Gavier-Widén1,2

Abstract Background: Prior to 2010, the lagoviruses that cause rabbit hemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus) and European brown hare syndrome (EBHS) in hares (Lepus spp.) were generally genus- specific. However, in 2010, rabbit hemorrhagic disease virus 2 (RHDV2), also known as Lagovirus europaeus GI.2, emerged and had the distinguishing ability to cause disease in both rabbits and certain hare species. The mountain hare (Lepus timidus) is native to Sweden and is susceptible to European brown hare syndrome virus (EBHSV), also called Lagovirus europaeus GII.1. While most mountain hare populations are found on the mainland, isolated populations also exist on islands. Here we investigate a mortality event in mountain hares on the small island of Hallands Väderö where other leporid species, including rabbits, are absent. Results: Post-mortem and microscopic examination of three mountain hare carcasses collected from early November 2016 to mid-March 2017 revealed acute hepatic necrosis consistent with pathogenic lagovirus infection. Using immunohistochemistry, lagoviral capsid antigen was visualized within lesions, both in hepatocytes and macrophages. Genotyping and immunotyping of the virus independently confirmed infection with L. europaeus GI. 2, not GII.1. Phylogenetic analyses of the vp60 gene grouped mountain hare strains together with a rabbit strain from an outbreak of GI.2 in July 2016, collected approximately 50 km away on the mainland. Conclusions: This is the first documented infection of GI.2 in mountain hares and further expands the host range of GI.2. Lesions and tissue distribution mimic those of GII.1 in mountain hares. The virus was most likely initially introduced from a concurrent, large-scale GI.2 outbreak in rabbits on the adjacent mainland, providing another example of how readily this virus can spread. The mortality event in mountain hares lasted for at least 4.5 months in the absence of rabbits, which would have required virus circulation among mountain hares, environmental persistence and/or multiple introductions. This marks the fourth Lepus species that can succumb to GI.2 infection, suggesting that susceptibility to GI.2 may be common in Lepus species. Measures to minimize the spread of GI.2 to vulnerable Lepus populations therefore are prudent. Keywords: Rabbit hemorrhagic disease, Lagovirus europaeus GI.2, RHDV2, Lepus timidus, Hare, Virus, Wildlife, Hepatitis

* Correspondence: [email protected] 1Department of Pathology and Wildlife Diseases, National Veterinary Institute (SVA), 751 89 Uppsala, Sweden 2Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences (SLU), Box 7028, 750 07 Uppsala, Sweden Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 2 of 12

Background hares. Confirmed cases in mountain hares have only oc- Pathogenic lagoviruses (Family Caliciviridae) cause curred in the southern half of the country where the hepatitis in leporids. The disease is referred to as rabbit ranges of European brown and mountain hares overlap hemorrhagic disease (RHD) or European brown hare [14, 15]. This suggests that the virus circulates mainly in syndrome (EBHS) depending on whether it affects rab- European brown hares and that mountain hares may be bits or hares. Viruses identified prior to 2010 are gener- spillover hosts [14]. ally considered to be genus-specific and do not cause In the early autumn of 2016, managers of a small is- clinical disease in animals less than five weeks of age. land nature reserve off the Swedish west coast began to Disease caused by rabbit hemorrhagic disease virus find carcasses of mountain hares. Here we describe an (RHDV) and its antigenic variant (RHDVa), also collect- outbreak of a pathogenic lagovirus in this island popula- ively known as Lagovirus europaeus GI.1 [1], is almost tion of mountain hares and provide evidence for the fur- exclusively confined to domestic and wild European rab- ther expansion of GI.2 host range. The outbreak bits (Oryctolagus cuniculus). Similarly, European brown occurred on an island without rabbits or European hares (Lepus europaeus) and other hare species including brown hares and we explore the epidemiological circum- mountain hares (Lepus timidus) are susceptible to EBHS stances of the outbreak, describe the pathology in moun- virus (EBHSV) [2], also called Lagovirus europaeus GII.1 tain hares, investigate the molecular epidemiology of [1], but not to GI.1 viruses. However, in 2010, RHDV2 GI.2, and discuss the implications of disease introduc- or RHDVb, also known as L. europaeus GI.2 [1], tions for isolated wildlife populations. emerged in France [3]. Differing significantly from the previously detected viruses, GI.2 has a broader age and Methods host range. GI.2 can cause clinical disease and death in Study site rabbits as young as 11 days old [4]. In addition to infect- Hallands Väderö (N 56° 26.6′, E 12° 33.7′) is a small, ing rabbits, GI.2 can also cause EBHS-like disease in the 310 ha island 3 km off the southwest coast of Sweden, Sardinian Cape hare (Lepus capensis mediterraneus)[5], accessible only by boat (Fig. 1). It is a nature reserve the Italian hare (Lepus corsicanus)[6] and the European with no permanent residents living on the island, but is brown hare [7–9]. host to approximately 50,000 visitors per year, primarily The mountain hare (Lepus timidus) is an arctic/sub- in the summer months. The island is also home to an in- arctic species that inhabits the tundra and taiga habitats troduced, managed mountain hare population and orga- of northern Europe and Asia [10]. While listed as a nized hunts are typically held 1–2 times per year. The species of least concern globally [11], certain regions in earliest documented introduction of hares occurred in Europe and Russia have been experiencing gradual 1857 from Godska Sandön, a Swedish island in the Baltic population declines [12]. This species is particularly Sea, and numerous restocking events have occurred prone to dramatic population crashes on islands [13]. In since then [16]. There are no European brown hares or Sweden, the mountain hare is a native species and it his- wild rabbits on the island, making the mountain hare torically ranged throughout the country. Isolated popula- the only leporid species present. tions of mountain hares also exist on many small Swedish islands because of relatively recent introduc- Animals tions [10]. While populations in the north are robust, In September 2016, managers of Hallands Väderö began mountain hare range and density have decreased in finding mountain hare carcasses. Remains of approxi- south and central Sweden since the beginning of the mately 15 animals were noted from September to twentieth century, and mountain hares have completely December 2016. In the beginning of November 2016, disappeared from the far south [12]. Postulated reasons the first dead hare (animal 2147) was collected, frozen for the decline include negative impacts from introduced and transported to the National Veterinary Institute European brown hares (Lepus europaeus), predation (SVA), Uppsala, Sweden for necropsy on November 17. pressure, competition with other herbivores, or land- Two other dead hares found at the beginning of scape change [12]. While competitive exclusion by, and December 2016 (animal 2381) and middle of March hybridization with, brown hares are considered primary 2017 (animal 1208) were frozen and transported to candidates for the decline of southern mountain hare SVA for examination on December 19, 2016 and March populations in Sweden, disease-mediated competition 29, 2017, respectively (Table 1). through, for example, GII.1 or Francisella tularensis may All three animals showed characteristic features of also play a role [12]. Although GII.1 can infect and cause Lepus timidus. Compared to Lepus europaeus, they were disease in both European brown hares and mountain smaller, had shorter ears and front limbs and the fore- hares, documented cases of EBHS in Sweden are geo- head was rounder [10]. The pelage was in varying stages graphically restricted to the range of European brown of moulting from grey-brown to grey-white (Fig. 2). The Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 3 of 12

Fig. 1 A map of the southern Sweden depicting cases of Lagovirus europaeus G1.2/RHDV2/b infection from which the vp60 gene sequences originated. The three mountain hares from Hallands Väderö (2147, 2381 and 1208) are represented by a triangle (▲), the rabbit from Falkenberg (1251) is depicted by a square (■) and other rabbits are shown with a circle (●). The base map is provided by Lantmäteriet, Sweden under the open data license CC0 animal found in November had the greatest proportion samples of two of the hares (2147 and 1208). DNA was of brown in its pelage and the animal in March was al- extracted with the EasySpin Genomic DNA Minipreps most completely grey-white. To confirm species identity, Tissue Kit (Citomed, Portugal) following manufacturer’s molecular analyses were also carried out on liver instructions. When considered together, the nuclear Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 4 of 12

Table 1 Mountain hares (Lepus timidus) found dead or hunted on Hallands Väderö, Sweden Hare identification number Found dead (D) or hunted (H) Date collected Sexa Ageb Body condition 2147 D November 2016 F Y Poor 2381 D December 2016 F A Normal 1208 D March 2017 M U Emaciated 123 H January 14, 2017 M NE Normal 124 H January 14, 2017 M NE Normal 126 H January 14, 2017 M NE Normal 127 H January 14, 2017 F A Normal 2539 H September 21, 2017 NE NE Normal 2540 H September 21, 2017 NE NE Normal aF female, M male, NE not examined bY young of year, A adult, U unable to determine NE, not examined genes uncoupling protein 2 (upc2) and immunoglobulin infection in these apparently healthy animals during (n = heavy constant gamma (ighg) and the mitochondrial 4, January) and after (n = 2, September) the outbreak. gene cytochrome b (cytb) distinguish L. timidus from Fresh liver and duodenum samples were collected from the other leporid species. Amplification of these markers animals hunted in January 2017 and frozen at − 20 °C. was carried out using the primers described in [17, 18]; Formalin-fixed tissues from these animals included liver, PCR conditions are available upon request. After purifi- spleen, duodenum, jejunum, ileum, sacculus rotundus and cation, PCR products were sequenced on an automatic cecal appendix. In September 2017, fresh and sequencer ABI PRISM 310 Genetic Analyzer (PE Ap- formalin-fixed liver were collected. plied Biosystems, USA) using the amplification primers. Sequences were submitted to the publicly available Gen- Bank database (https://www.ncbi.nlm.nih.gov/genbank) Post-mortem and microscopic examination and bacteriology under the accession numbers MH107277–82. Genetic All three fallen animals were examined by necropsy. distances between the sequences of 2147 and 1208 and Nutritional condition was determined according to the other lagomorphs were calculated for each gene in following criteria: emaciated (visible muscle atrophy, ab- MEGA6 [19] using the following parameters: p-distance sence of visible fat stores), poor (scant to no visible in- method, 500 replicates, pairwise deletion. ternal fat stores, but no visible muscle atrophy), normal Management of the mountain hare population on (at a minimum, fat stores visible in mesentery and Hallands Väderö included a hunt in January and around kidneys). A suite of internal organs including September 2017. Sampling of these hunted animals liver, spleen, lung, kidney, bone marrow, brain, small in- provided an opportunity to screen for evidence of GI.2 testine, colon, heart, trachea, salivary gland, nasal

Fig. 2 A mountain hare (Lepus timidus) that died of Lagovirus europaeus GI.2/RHDV2/b infection on the island of Hallands Vädero, Sweden. a Characteristic external features include a grey-white winter pelage. b Post-mortem findings. The liver shows pallor and enhanced reticular pattern (arrow). Distal lung lobes are consolidated because of nematode parasitism (arrowhead) Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 5 of 12

mucosa and skin were fixed in 10% neutrally buffered for immunological typing of the lagovirus. Briefly, typing formalin for microscopic examination. Not all tissues analysis was performed using the sandwich ELISA de- were collected from all animals. Liver was frozen at − 20 scribed in the OIE Terrestrial Manual for RHD (2016) °C and − 70 °C for additional analyses. [26]. Four pools of monoclonal antibodies specific for Formalin-fixed tissues from the found dead and GI.1b-d/RHDV [22], GI.1a/RHDVa [27], GI.2/RHDV2 [5] hunted hares were processed and embedded in paraffin and GII.1/EBHSV [15] were used. Samples (10% liver ho- for microscopic examination. Sections 3 to 4 μm thick mogenates) were tested at the dilution 1/5 and 1/30 and were stained with Mayer’s hematoxylin and eosin [20]. classified as positive when the OD of the sample exceeded Liver sections also were stained with von Kossa to that of the negative control by at least 0.15 at dilution 1/5. visualize calcium deposits [21]. A more detailed description of the methods is provided in Even though no organisms were seen microscopically, Neimanis et al. [24]. the mild to moderate heterophilic infiltration observed in the liver of 2147 prompted the submission of liver Genotyping and phylogenetic analyses from this animal to the Department of Microbiology, Based on gross and microscopic findings typical for National Veterinary Institute, Uppsala, Sweden for rou- pathogenic lagovirus infection in the three hares found tine aerobic bacterial culture. Samples were inoculated dead, various molecular analyses were carried out on onto blood agar plates containing 5% horse blood and liver samples to confirm the presence of a lagovirus and bromocresol purple lactose agar plates and held at 37 °C to genotype the virus. For hares 1208 and 2147, samples under aerobic conditions. Plates were inspected for were first analyzed for the presence of GI and GII.1 with growth at 24 and 48 h after inoculation. two nested PCRs using primers and methods as de- scribed in Bascuñana et al. [25]. In both cases, GI but Immunohistochemistry not GII.1 RNA was detected. Because this nested PCR Immunohistochemistry was used to detect the presence of does not differentiate between GI.1 and GI.2 viruses lagovirus antigen in the formalin-fixed and [24], liver samples from these two hares and from 2381 paraffin-embedded tissues using a panlagovirus antibody were analyzed by GI.2-specific RT-qPCR as described in cocktail of 3H6 + 6G2 IgG1 mouse monoclonal antibodies Neimanis et al. [24]. To screen for the presence of GI.2, [22] kindly provided by the OIE Reference Laboratory for liver samples from all six hunted hares were also ana- RHD, Brescia, Italy. Immunohistochemical staining was lyzed by this same GI.2-specific RT-qPCR. performed on liver, spleen, lung, bone marrow and kidney For samples found to contain GI.2 RNA by RT-qPCR, from the three fallen hares and on the liver and small in- sequencing of the entire vp60 gene following methods in testine of hares hunted in January. Methods, antibodies Neimanis et al. [24] was carried out. In 2016 and 2017, a and reagents are described in detail in Neimanis et al. [23] concurrent, widespread outbreak of GI.2 occurred in with the following modifications. After treatment with wild and domestic rabbits in the southern half of hydrogen peroxide, sections were treated with proteinase Sweden [24]. To gain insight into the origins of GI.2 in K (Dako Proteinase K, Agilent Technologies Sweden AB, mountain hares on Hallands Väderö, the entire vp60 Kista, Sweden) for six minutes to demask antigens. gene was also sequenced from the livers of ten additional Visualization of bound antibodies was facilitated with ei- rabbits known to have died from GI.2 infection for ther the chromagen diaminobenzidine (DAB) (Dako Li- phylogenetic comparison with hare strains. Rabbit sam- quid DAB+ Substrate Chromagen System, Agilent ples were selected to represent the broad spatial and Technologies Sweden AB, Kista, Sweden) or the chroma- temporal range of the outbreak. Locations from where gen 3-amino-9-ethylcarbazole (AEC) (Dako AEC+ High these 10 rabbits, the three fallen mountain hares and an Sensitivity Substrate Chromagen, Agilent Technologies additional 11 rabbits previously reported with GI.2 in Sweden AB, Kista, Sweden). The liver from a rabbit con- Sweden [24] originated are presented in Fig. 1. All vp60 firmed to be infected with GI.2 by PCR, sequencing and gene sequences generated in this study were deposited immunological characterization [24] and/or the liver from in GenBank under accession numbers MH341501–13. a European brown hare confirmed to be infected with To identify homologous sequences in GenBank, a GII.1 by nested PCR [25] was used as the positive tissue Basic Local Alignment Search Tool (BLAST, http:// control while the liver from a healthy rabbit and/or www.ncbi.nlm.nih.gov/BLAST/) was used on the vp60 European brown hare (negative for lagoviruses by PCR) gene sequences from the three fallen hares. Additionally, served as the negative tissue control. evolutionary relationships between the mountain hare viruses and other lagoviruses were explored. Sequences Immunological virus typing were aligned using the Clustal W algorithm before Liver samples from all three fallen hares were submitted building phylogenetic trees using MEGA6: Molecular to the OIE Reference Laboratory for RHD in Brescia, Italy Evolutionary Genetics Analysis [19]. The best-fit Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 6 of 12

nucleotide substitution model (GTR + G + I) was deter- and the spleen was discoloured dark red in hares 2147 mined with the same software. The final alignment in- and 2381. All animals had soft, unformed feces in the cluded vp60 gene sequences generated for this study rectum and in 2381, the cecal appendix and sacculus (from the three mountain hares and 10 rabbits) and pub- rotundus were diffusely dark red. There was mild to licly available GI.2 sequences from Europe and Australia, moderate consolidation of the distal margins of the cau- including 11 previously published sequences from dal lung lobes consistent with nematode infestation in Sweden from 2013 to 2016 [24]. GI.1, GI.4 and GII.1 se- all animals (Fig. 2) and few to more frequent 1 × 2 mm quences, as well as MRCV (a lagovirus with variable beige, oblong foci consistent with coccidial organisms pathogenicity [28]) and other sequences not assigned to were seen in the small intestine of hares 2147 and 1208. a genotype, were included in the analysis, producing a final dataset of n = 364. A list of all sequences used is Microscopic pathology provided as supplementary material in Additional file 1. All fallen hares had been previously frozen and were Trees were built using the Maximum likelihood (ML) moderately to severely autolyzed. However, liver lesions method and statistical support for the nodes was esti- could still be detected. All animals displayed acute ne- mated using 2000 bootstrap replicates. crosis and/or apoptosis of individual hepatocytes charac- terized by hypereosinophilic, hyaline cytoplasm, cell Results shrinkage, rounding up and dissociation from the hep- Sex, age and nutritional condition of the mountain hares atic plate (Fig. 3). Affected hepatocytes were observed are presented in Table 1. Body condition of the fallen throughout the lobule. In hares 2147 and 1208, small hares ranged from normal to emaciated whereas the scattered, focal areas of lytic necrosis were also seen. hunted hares were all in normal nutritional condition. Additionally, dark grey-basophilic, irregular intracyto- Age was not assessed for the majority of the hunted ani- plasmic granules confirmed to be mineral using von mals and sex was not determined for animals hunted in Kossa histochemical stain were observed in scattered September 2017. (2147) to frequent (1208) hepatocytes (Fig. 3). In all hares, mildly to moderately increased numbers of in- Hares found dead flammatory cells, most consistent with heterophils, were Molecular species identification observed in sinusoids and occasionally surrounding indi- The obtained sequences for ucp2, ighg-hinge and cytb vidual hepatocytes (Fig. 3). There also was mild (2147 were visually inspected and aligned with publicly avail- and 1208) to moderate (2381) microvesiculation of hepa- able sequences for lagomorphs. Only species with infor- tocytes consistent with lipid. Hare 2381 had a mild mation for at least two markers were considered. trematode infestation evidenced by scattered trematode Genetic distances indicate that our samples are closest eggs observed within larger bile ducts. to three hare species L. timidus, L. arcticus and L. othus Microscopic examination of the lungs and small intes- and clearly distinct from the European brown hare. tine confirmed granulomatous pneumonia from lung- Despite the lack of resolution, we confirm the species as worm infection and intestinal coccidiosis, respectively. being L. timidus, which is consistent with the morpho- Autolysis precluded further interpretation of other tissues. logical characterization and species geographic range (L. arcticus inhabits the coastal regions of Greenland, north- ern Quebec, northern Manitoba, Arctic islands and Immunohistochemistry western Newfoundland; L. othus ranges from west to Viral antigen was detected in the liver of all three fallen southwest Alaska). The three arctic hare species (L. timi- hares (Fig. 3). In 2147 and 2381, the antigen was primar- dus, L. arcticus and L. othus) are closely related and their ily confined to hepatocytes as fine intracytoplasmic stip- status as independent species has been critically dis- pling or concentrated at the cell membrane, although cussed [29, 30]. occasional intranuclear staining also was seen (Fig. 3). In 1208, staining frequently was seen within the cytoplasm Gross pathology of both hepatocytes and Kupffer cells (Fig. 3), and very All three animals found dead and examined by necropsy rarely within hepatocellular nuclei. Immunostaining was had subtle to moderate liver lesions in which the liver not detected in the negative controls. was mildly to moderately discoloured (light brown to Immunohistochemical evaluation of the spleen of 2147 orange-pink), had an accentuated lobular pattern in the and 2381 was hindered by autolysis and artifact. How- parenchyma, and was friable (Fig. 2). All three animals ever, within the spleen of 1208, viral antigen was clearly also displayed mild to moderate pulmonary congestion and coarsely clumped within the cytoplasm of macro- and multifocal to coalescing hemorrhage and/or edema. phages (Fig. 3). Poor tissue quality precluded interpret- There was mild to moderate splenomegaly in all animals ation of immunohistochemistry for remaining tissues. Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 7 of 12

Fig. 3 Microscopic lesions and viral antigen localization in mountain hares (Lepus timidus) that died of Lagovirus europaeus GI.2/RHDV2/b infection. a Acute hepatocellular apoptosis and necrosis. Dead cells are denoted with an arrow and they frequently are surrounded by heterophils. The portal region is denoted by an asterisk. b Acute hepatocellular apoptosis and necrosis (arrows). Arrowhead denote hepatocellular calcification and the asterisk marks the portal area. Insert: von Kossa histochemical stain with calcium granules stained black. c Immunohistochemical visualization of viral capsid antigen (brown) in the liver of hare 2147. Arrows denote hepatocytes with both intracytoplasmic and intranuclear staining. d Immunohistochemical visualization of viral capsid antigen (brown) in the liver of hare 1208. Intracytoplasmic staining of hepatocytes (arrow) and Kupffer cells (arrowhead). e Immunohistochemical visualization of viral capsid antigen (brown) in the spleen of hare 1208. Arrows indicate intracytoplasmic staining of macrophages in the red pulp. f Negative immunoglobulin control of the liver of 1208 Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 8 of 12

Bacteriology immunohistochemistry was not performed on the two No bacterial infection was identified in the liver of hare hares hunted in September 2017. 2147. Immunological virus typing Hunted hares In all three fallen hares, the lagovirus in the liver was Pathology and immunohistochemistry identified as GI.2 by the specific pool of MAbs directed No significant lesions were observed grossly in the against GI.2. Results for the other pools of MAbs spe- hunted hares. Microscopically, no lesions consistent with cific for GI.1b-d, GI.1a and GII.1 were negative. Add- pathogenic lagoviruses were seen in the liver. In hares itionally, the high OD values registered at the dilution of 126 and 127, hilar bile ducts contained cross-sections of 1/30 are consistent with acute lagovirus infection and adult trematodes and/or thick-walled yellow-brown cause of the death of the hares. trematode eggs that were accompanied by mild to moder- ate periductal eosinophilic and lymphoplasmacytic inflam- Molecular detection and phylogenetic analysis mation. Small intestine only was available for examination A 127-base pair fragment from the vp60 capsid gene of in hares hunted in January 2017. Although scattered intra- GI.2 was amplified in large quantities in the liver of all luminal nematodes were observed in hares 124, 126 and three hares that were found dead. The threshold cycle 127, the intestinal tissue was unremarkable. values ranged from 15.24 to 15.96. RT-qPCR did not de- No viral antigen was detected in the liver or small tect GI.2 in any of the livers of the six hunted hares. intestine of hares hunted in January 2017 and Complete vp60 gene sequences were determined for all

Fig. 4 Phylogenetic analysis of complete vp60 gene sequences of Lagovirus europaeus GI.2/RHDV2/b obtained from mountain hares (Lepus timidus) and European rabbits (Oryctolagus cuniculus) in Sweden in relation to other publicly available lagovirus vp60 gene sequences. The tree was generated in MEGA6 [19] using the Maximum Likelihood method and GTR + G + I nucleotide substitution model. Bootstrap values ≤70% (of 2000 replicates) are indicated at the nodes. A complete list of GenBank accession numbers used in the analysis is provided in Additional file 1 Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 9 of 12

three fallen hares and 10 rabbits that died of RHD in and we support the proposal by Le Gall-Reculé et al. [9] other areas of Sweden in 2016 (Genbank accession num- for a new name. While ‘hare hemorrhagic disease’ makes bers MH341501–13). reference to the equivalent disease in rabbits (RHD), A BLAST comparison of the vp60 gene sequences hemorrhage and disseminated intravascular coagulation from the three mountain hares with publicly available (DIC) are not prominent features of pathogenic lago- sequences revealed the closest identity with GI.2. More virus infections in hares ([32, 34], this study). We there- specifically, they showed a maximum identity of 98% at fore support hare lagovirus disease or hare lagoviral the nucleotide level to the 16PLM1 isolate (GenBank ac- hepatitis as an alternate name for viral hepatitides of cession number MF407653), a non-recombinant GI.2 hares caused by different pathogenic lagoviruses. strain from La Palma, Canary Islands, Spain, collected in This outbreak of GI.2 in mountain hares is also signifi- 2016 [31] (comparison performed March 20, 2018). cant because it represents an expansion of GI.2 host Phylogenetic analyses grouped all three mountain hare range. GI.2 differs notably from previously described GI strains together, along with other strains collected in viruses because of its broader host range. Clinical disease 2016–2017 for this study (Fig. 4). Strain 1251 came from from GI.1 viruses is almost exclusively restricted to the a GI.2 outbreak in rabbits in Falkenberg and was col- European rabbit, with the exception of GI.1 infection de- lected at the end of July 2016 on the adjacent mainland scribed in two Iberian hares (Lepus granatensis)[35]. In approximately 50 km away (Fig. 1). The other three contrast, GI.2 can be pathogenic for European rabbits, strains that clustered near the hare strains (1318, 8015 but also for the Sardinian Cape hare [5], the Italian hare and 1362) came from GI.2 outbreaks in southern main- [6], the European brown hare [7–9] and now the moun- land Sweden in July and August 2016. tain hare. Certain hare species are proposed to be more susceptible to GI.2 infection than others [6] and this Discussion may reflect different species-specific host factors such as The detection of lagoviral antigen associated with lesions glycan expression for viral attachment [36]. However, of acute hepatitis, coupled with immunotyping and mo- the epidemiological context may also play a role. For ex- lecular detection of GI.2 in the liver, conclusively show ample, the broad and rapid dispersion of GI.2 in rabbits that all three fallen mountain hares on Hallands Väderö in France and Sardinia when compared to mainland Italy died of acute viral hepatitis caused by GI.2. This is sig- is thought to be related to the widespread presence of nificant because, prior to this study, mountain hares wild rabbits in the former areas versus sparse and patch- were only known to be susceptible to GII.1, the patho- ily distributed wild rabbit populations in continental genic lagovirus of hares causing EBHS. The gross and Italy [3]. Sporadic infection of European brown hares microscopic pathology caused by GI.2 in mountain hares with GI.2 in mainland Italy [7] compared to large-scale and tissue distribution of viral antigen mimics that of outbreaks of GI.2 in European brown hares in France [9] GII.1 infection in this species. Consistent gross lesions may also reflect differences in the presence and densities include a lighter coloured and friable liver, a dark red, of sympatric wild rabbits and thus environmental virus congested and often enlarged spleen and pulmonary loads. To date, although mountain hares range throughout congestion and edema. Microscopically, periportal to much of Sweden, GI.2 infection has only been detected in massive hepatic necrosis has been described for GII.1 in- mountain hares on the small island of Hallands Väderö. It fections in mountain hares [32], and in this study, necro- is unclear if all mountain hares are equally susceptible or sis was classified as massive. Other lesions in these three if this particular mountain hare population was somehow hares that are consistent with GII.1 infections include predisposed to GI.2 infection. Genetic isolation, concur- areas of lytic necrosis, inflammation, hepatocellular fatty rent parasitic infections (found in all three dead hares and degeneration and mineralization of hepatocytes. Interest- in three of four hunted hares that were examined micro- ingly, mineralization seems to be a phenomenon of lago- scopically) and the poor nutritional condition (seen in two viral hepatitis in hares, irrespective of the causative of the three dead hares and observed in hares hunted in virus, and is not a feature commonly associated with October 2016, B. Gunnarsson, pers. comm) may have RHD [33]. Viral antigen in the cytoplasm and nuclei of made this population of animals more prone to GI.2 infec- hepatocytes and cytoplasm of Kupffer cells was de- tion. Elucidation of the susceptibility of other mountain scribed both for GII.1 [31] and GI.2 in mountain hares hare populations to GI.2 requires further field and/or ex- (this study). Gross and microscopic lesions can still be perimental research. considered characteristic of infection with a pathogenic Epidemiological and molecular data from the majority lagovirus, but further analyses are required to identify of previously reported GI.2 outbreaks in hares support a the lagovirus responsible. Just as for other hare species link to concurrent local or regional outbreaks in European that succumb to GI.2 infection, EBHS is no longer an rabbits [5, 6, 8, 9]. Outbreaks typically were connected in adequate name for lagoviral hepatitis in mountain hares time and/or space with outbreaks in rabbits and, when Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 10 of 12

data were available, the GI.2 virus in affected hares was competition from brown hares through, for example, most closely related to those strains locally circulating in GII.1, has been proposed as a possible contributing factor rabbits [6, 9]. This suggests that while hares are suscep- [12]. It is now clear that mountain hares can also succumb tible to infection, they require spillover of GI.2 from the to GI.2 infection. Wild rabbit distribution also overlaps primary maintenance host, the rabbit, to initiate new out- with areas where mountain hares are in decline. The ar- breaks in hares. The findings presented here provide fur- rival of GI.2 to Sweden and subsequent large-scale epizo- ther support for this hypothesis. Although there are no otics in rabbits may now pose an additional threat to rabbits on the island from which initial spillover of infec- sympatric mountain hares. The impact of GI.2 on isolated tion could have originated, there was a concurrent, wide- island populations of mountain hares may be even more spread outbreak of GI.2 in wild and domestic rabbits in significant. Island populations of mountain hares are par- the southern half of Sweden in 2016 and 2017, including ticularly vulnerable to population crashes [13]. Factors re- the adjacent mainland. The GI.2 vp60 gene sequences sponsible for crashes such as food availability are often from mountain hares grouped together with a sequence multifactorial and declines are often set off by a stochastic collected from an outbreak in rabbits at the end of July event [13]. While extreme weather and appearance of a 2016 on the adjacent mainland in Falkenberg (Fig. 1), predator have been cited as stochastic events, we propose approximately 50 km away. The other vp60 gene se- that the sudden introduction of GI.2 could exert the same quences from Sweden represent outbreaks of GI.2 in rab- effect and trigger a decline. Continued monitoring of the bits that were more distant from the outbreak on hare population on Hallands Väderö is needed to assess Hallands Väderö in time and/or space (Fig. 1). the longer-term significance of GI.2 introduction for this Pathogenic GI viruses can be readily transmitted indir- population. No evidence of GI.2 infection was found in six ectly via contaminated material and transfer can be facil- hares hunted in January and September 2017 and, with itated by insects, scavengers and people [37]. The exact the exception of a juvenile that died of intestinal coccidi- mode of virus introduction onto Hallands Väderö is not osis, no further reports of dead hares from the island have known, but introduction by the 50,000 people who visit been received to date. GI.2 has spread quickly and widely the island every year, by insects or by scavenging birds throughout Europe and beyond, and, as evidenced by this are all possibilities. Once the virus arrived on the island, outbreak, the virus can be introduced to islands and circu- GI.2 circulated for at least 4.5 months based on necropsy late among hares in the absence of rabbits. Awareness of findings, and six months based on observations of dead this potential risk coupled with actions that minimize un- hares in the field, all in the absence of rabbits. GI.2 was intended introductions of GI.2 by humans through, for ex- either introduced once to the island and then persisted ample, contaminated footwear, can help mitigate exposure for the duration of the outbreak or it was repeatedly in- of other island populations of mountain hares to GI.2. troduced. VP60 gene sequences of all three hares group very closely together, with only one, synonymous nucleo- Conclusions tide substitution between the two hares found dead in We demonstrate for the first time that GI.2 can infect 2016 and four synonymous and one non-synonymous and cause mortality in mountain hares. These findings nucleotide substitutions in the hare found dead in 2017. add a new species to the host range of GI.2. Further in- This lends support to a single, rather than multiple, vestigation of the mechanisms responsible for the ex- introduction, but sequencing of the whole genome of panded host range of GI.2 compared to GI.1 viruses these hares and additional rabbit samples followed by could provide valuable insight into host species jumps. spatial and temporal analyses are needed to further sub- Lesions and virus tissue distribution of GI.2 infection in stantiate this hypothesis. GI viruses are hardy and can mountain hares mimic those of GII.1 infection, therefore persist in the environment for months in organic mater- typing of the virus must be performed to identify the ial [38]. GI.2 may have circulated within the mountain lagovirus responsible for a given mortality event. The hare population during the outbreak in the absence of mortality event in mountain hares lasted at least 4.5 rabbits and/or the introduced virus may have persisted months in the absence of rabbits, but because environ- in the environment (e.g. in feces or hare carcasses), mental persistence and multiple introductions cannot be causing re-infections of hares over the period of the out- excluded, further research is required to determine if break. Therefore, the ability of mountain hares to serve mountain hares are competent reservoir hosts. Phylo- as competent maintenance hosts for GI.2 in the absence genetic analyses support virus introduction from a con- of other leporids presently is unknown and requires add- current, large-scale GI.2 outbreak in rabbits on the itional investigation. adjacent mainland and demonstrates the relative ease Mountain hare populations are declining in the southern with which GI.2 can spread. Finally, mountain hares rep- half of Sweden, coinciding geographically with where they resent the fourth Lepus species that is susceptible to are sympatric with brown hares [12]. Disease-mediated GI.2. This raises the possibility that most or even all Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 11 of 12

Lepus species may be susceptible. Not only should inci- Author details 1 dents of morbidity and mortality in other Lepus species Department of Pathology and Wildlife Diseases, National Veterinary Institute (SVA), 751 89 Uppsala, Sweden. 2Department of Biomedical Sciences and sympatric with wild European rabbits or in contact with Veterinary Public Health, Swedish University of Agricultural Sciences (SLU), domestic rabbits be evaluated with respect to GI.2, but Box 7028, 750 07 Uppsala, Sweden. 3Department of Microbiology, National 4 exposure to European rabbits should be minimized for Veterinary Institute (SVA), 751 89 Uppsala, Sweden. CIBIO/InBio, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, vulnerable Lepus species and populations. Campus Agrário de Vairão, 4485-661 Vairão, Portugal. 5Department of Anatomy and Unit for Multidisciplinary Research in Biomedicine (UMIB), Additional file Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal. 6Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, R. Campo Alegre s/n, 4169-007 Porto, Portugal. Additional file 1: GenBank accession number, sequence name and 7Instituto de Investigação e Formação Avançada em Ciências e Tecnologias Lagovirus europaeus genotype (after [1]) for the vp60 gene sequences da Saúde (CESPU), Gandra, Portugal. used in the phylogenetic analysis. (XLSX 22 kb) Received: 28 May 2018 Accepted: 12 November 2018 Abbreviations BLAST: Basic Local Alignment Search Tool; EBHS: European brown hare syndrome; EBHSV: European brown hare syndrome virus; MAb: Monoclonal References antibody; ML: Maximum likelihood; OD: Optical density; PCR: Polymerase chain 1. Le Pendu J, Abrantes J, Bertagnoli S, Guitton JS, Le Gall-Reculé G, Lopes AM, reaction),; RHD: Rabbit hemorrhagic disease; RHDV: Rabbit hemorrhagic disease Marchandeau S, Alda F, Almeida T, Célio AP, Bárcena J, Burmakina G, Blanco virus; RT-qPCR: Reverse transcription quantitative polymerase chain reaction E, Calvete C, Cavadini P, Cooke B, Dalton K, Delibes Mateos M, Deptula W, Eden JS, Wang F, Ferreira CC, Ferreira P, Foronda P, Gonçalves D, Gavier- Acknowledgements Widén D, Hall R, Hukowska-Szematowicz B, Kerr P, Kovaliski J, Lavazza A, The authors thank Bo Gunnarsson for submission of the dead hares, Caroline Mahar J, Malogolovkin A, Marques RM, Marques S, Martin-Alonso A, Bröjer, Holly Cedervind, Bertil Malmsten and Carl-Gustav Thulin for help with Monterroso P, Moreno S, Mutze G, Neimanis A, Niedzwiedzka-Rystwej P, sample and data collection and Linda Svensson for assistance with Fig. 1. Peacock D, Parra F, Rocchi M, Rouco C, Ruvoën-Clouet N, Silva E, Silvério D, Lorenzo Capucci, OIE Reference laboratory for RHD, generously provided the Strive T, Thompson G, Tokarz-Deptula B, Esteves P. Proposal for a unified monoclonal antibodies used in the immunohistochemistry and performed classification system and nomenclature of lagoviruses. J Gen Virol. 2017; the immunotyping. 98(7):1658–66. https://doi.org/10.1099/jgv.0.000840. 2. Gavier-Widén D, Mörner T. Epidemiology and diagnosis of the European Funding brown hare syndrome in Scandinavian countries: a review. Rev Sci Tech. Funding was provided by the Swedish Environmental Protection Agency, 1991;10(2):453–8. and the Research Council FORMAS (contract 221–2014-1841) as part of the 3. Le Gall-Reculé G, Lavazza A, Marchandeau S, Bertagnoli S, Zwingelstein F, ECALEP research consortium funded by the EU Animal Health and Welfare Cavadini P, Martinelli N, Lombardi G, Guerin J-L, Lemaitre E, Decors A, ERA-Net (ANIHWA). Fundação para a Ciência e Tecnologia (FCT) supported Boucher S, Le Normand B, Capucci L. Emergence of a new lagovirus related the Post-doctoral grant of Ana M. Lopes (SFRH/BPD/115211/2016) and the to rabbit haemorrhagic disease virus. Vet Res. 2013;44. https://doi.org/10. FCT Investigator grants of Joana Abrantes (IF/01396/2013) and Pedro J. 1186/1297-9716-44-81. Esteves (IF/00376/2015). The funding bodies had no role in the design of the 4. Dalton KP, Nicieza I, Abrantes J, Esteves PJ, Parra F. Spread of new variant study, in the collection, analysis and interpretation of the data or in the RHDV in domestic rabbits on the Iberian Peninsula. Vet Microbiol. 2014; writing of the manuscript. 169(1–2):67–73. https://doi.org/10.1016/j.vetmic.2013.12.015. 5. Puggioni G, Cavadini P, Maestrale C, Scivoli R, Botti G, Ligios C, Le Gall- Availability of data and materials Reculé G, Lavazza A, Capucci L. The new French 2010 rabbit hemorrhagic The data supporting the conclusions of this article are included within the disease virus causes an RHD-like disease in the Sardinian cape hare (Lepus article and its additional file and genome sequences are publicly available in capensis mediterraneus). Vet Res. 2013;44. https://doi.org/10.1186/1297- the GenBank database (https://www.ncbi.nlm.nih.gov/genbank) under the 9716-44-96. accession numbers MH107277–82 and MH341501–13. 6. Camarda A, Pugliese N, Cavadini P, Circella E, Capucci L, Caroli A, Legretto M, Mallia E, Lavazza A. Detection of the new emerging rabbit haemorrhagic Author’s contributions disease type 2 virus (RHDV2) in Sicily from rabbit (Oryctolagus cuniculus) AN conceived the study, AN, HA and ULP acquired the samples and data and and Italian hare (Lepus corsicanus). Res Vet Sci. 2014;97(3):642–5. https://doi. performed the laboratory work, AN, HA, AL, JA, SZ, PE and DGW analyzed and org/10.1016/j.rvsc.2014.10.008. interpreted the data, AN wrote the paper, HA, ULP, AL, JA, SZ, PE and DGW 7. Velarde R, Cavadini P, Neimanis A, Cabezon O, Chiari M, Gaffuri A, Lavin S, revised the paper and all authors approved the final manuscript. Grilli G, Gavier-Widén D, Lavazza A, Capucci L. Spillover events of infection of brown hares (Lepus europaeus) with rabbit haemorrhagic disease type 2 Ethics approval and consent to participate virus (RHDV2) caused sporadic cases of an European brown hare syndrome- Samples from hunted hares were collected opportunistically during the like disease in Italy and Spain. Transbound Emerg Dis. 2017;64(6):1750–61. normal, regulated game hunt conducted by licensed Swedish hunters. No https://doi.org/10.1111/tbed.12562. animals were killed for the purpose of this study and no live animals or 8. Hall RN, Peacock DE, Kovaliski J, Mahar JE, Mourant R, Piper M, Strive T. tissues from live animals were used, therefore no permit from an animal Detection of RHDV2 in European brown hares (Lepus europaeus) in ethics committee was required for this study. Australia. Vet Rec. 2017;180(5):121. https://doi.org/10.1136/vr.104034. 9. Le Gall-Reculé G, Lemaitre E, Bertagnoli S, Hubert C, Top S, Decors A, Consent for publication Marchandeau S, Guitton J-S (2017) Large-scale lagovirus disease outbreaks Not applicable. in European brown hares (Lepus europaeus) in France caused by RHDV2 strains spatially shared with rabbits (Oryctolagus cuniculus). Vet Res 48 (1): Competing interests 70. doi:https://doi.org/10.1186/s13567-017-0473-y. – The authors declare they have no competing interests. 10. Angerbjorn A, Flux JEC. Lepus timidus. Mamm Species. 1995;(495):1, 1 1. 11. IUCN (2008) Lepus timidus. http://www.iucnredlist.org/details/11791/0. Accessed 6 May 2018. Publisher’sNote 12. Thulin C-G. The distribution of mountain hares Lepus timidus in Europe: a Springer Nature remains neutral with regard to jurisdictional claims in challenge from brown hares L. europaeus? Mammal Rev. 2003;33(1):29–42. published maps and institutional affiliations. https://doi.org/10.1046/j.1365-2907.2003.00008.x. Neimanis et al. BMC Veterinary Research (2018) 14:367 Page 12 of 12

13. Angerbjörn A. Proximate causes of mountain hare population crashes: a 35. Lopes AM, Marques S, Silva E, Magalhaes MJ, Pinheiro A, Alves PC, Jl P, review. Finn Game Res. 1983;41:29–38. Esteves PJ, Thompson G, Abrantes J. Detection of RHDV strains in the 14. Gavier-Widén D, Mörner T. Descriptive epizootiological study of European Iberian hare (Lepus granatensis): earliest evidence of rabbit lagovirus cross- brown hare syndrome in Sweden. J Wildl Dis. 1993;29(1):15–20. species infection. Vet Res. 2014;45:94. 15. Lopes AM, Capucci L, Gavier-Widén D, Le Gall-Reculé G, Brocchi E, Barbieri I, 36. Lopes AM, Breiman A, Lora M, le Moullac-Vaidye B, Galanina O, Nyström K, Quemener A, Le Pendu J, Geoghegan JL, Holmes EC, Esteves PJ, Abrantes J. Marchandeau S, Le Gall-Reculé G, Strive T, Neimanis A, Bovin NV, Ruvoën- Molecular evolution and antigenic variation of European brown hare Clouet EPJ, Abrantes J, Le Pendu J. Host specific glycans are correlated with syndrome virus (EBHSV). Virology. 2014;468:104–12. https://doi.org/10.1016/j. susceptibility to infection by lagoviruses, but not with their virulence. J Virol. virol.2014.08.002. 2018;92:e01759–17. https://doi.org/10.1128/JVI.01759-17. 16. Isacson C-G. Hallands Väderö och jakten på ön. In: Isacson international AB. 37. Abrantes J, van der Loo W, Le Pendu J, Esteves PJ. Rabbit haemorrhagic Sweden: Gothenburg; 1993. disease (RHD) and rabbit haemorrhagic disease virus (RHDV): a review. Vet 17. Melo-Ferreira J, Alves PC, Freitas H, Ferrand N, Boursot P. The genomic Res. 2012;43. https://doi.org/10.1186/1297-9716-43-12. legacy from the extinct Lepus timidus to the three hare species of Iberia: 38. Henning J, Meers J, Davies PR, Morris RS. Survival of rabbit haemorrhagic contrast between mtDNA, sex chromosomes and autosomes. Mol Ecol. disease virus (RHDV) in the environment. Epidemiol Infect. 2005;133(4):719– 2009;18:2643–58. 30. https://doi.org/10.1017/s0950268805003766. 18. Pinheiro A, Woof JM, Almeida T, Abrantes J, Alves PC, Gortázar C, Esteves PJ. Leporid immunoglobulin G shows evidence of strong selective pressure on the hinge and CH3 domains. Open Biol. 2014;4(9):140088. https://doi.org/10. 1098/rsob.140088. 19. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9. 20. Bancroft JD, Cook HC. Manual of histological techniques. London, England: Churchill Livingstone; 1984. 21. Lillie RD. Histopathologic technic and practical histochemistry. 3rd ed. New York: Blakiston Division, McGraw-Hill; 1965. 22. Capucci L, Frigoli G, Rønshold L, Lavazza A, Brocchi E, Rossi C. Antigenicity of the rabbit hemorrhagic disease virus studied by its reactivity with monoclonal antibodies. Virus Res. 1995;37:221–38. 23. Neimanis A, Larsson Pettersson U, Huang N, Gavier-Widén D, Strive T. Elucidation of the pathology and tissue distribution of Lagovirus europaeus GI.2/RHDV2 (rabbit haemorrhagic disease virus 2) in young and adult rabbits (Oryctolagus cuniculus). Vet Res. 2018;49:46. https://doi.org/10.1186/s13567- 018-0540-z. 24. Neimanis AS, Ahola H, Zohari S, Larsson Pettersson U, Bröjer C, Capucci L, Gavier-Widén D. Arrival of rabbit haemorrhagic disease virus 2 to northern Europe: emergence and outbreaks in wild and domestic rabbits (Oryctolagus cuniculus) in Sweden. Transbound Emerg Dis. 2017. https://doi. org/10.1111/tbed.12650. 25. Bascuñana CR, Nowotny N, Belak S. Detection and differentiation of rabbit hemorrhagic disease and European brown hare syndrome viruses by amplification of vp60 genomic sequences from fresh and fixed tissue specimens. J Clin Microbiol. 1997;35(10):2492–5. 26. OIE Rabbit Haemorrhagic Disease.https://www.oie.int/fileadmin/Home/eng/ Health_standards/tahm/2.06.02_RHD.pdf. 2016. Accessed 6 May 2018. 27. McIntosh MT, Behan SC, Mohamed FM, Lu Z, Moran KE, Burrage TG, Neilan JG, Ward GB, Botti G, Capucci L, Metwally SA. A pandemic strain of calicivirus threatens rabbit industries in the Americas. Virol J. 2007;4. https:// doi.org/10.1186/1743-422x-4-96. 28. Bergin IL, Wise AG, Bolin SR, Mullaney TP, Kiupel M, Maes RK. Novel Calicivirus identified in rabbits, Michigan, USA. Emerg Infect Dis. 2009;15(12): 1955–62. https://doi.org/10.3201/eid1512.090839. 29. Waltari E, Cook JA. Hares on ice: phylogeography and historical demographics of Lepus arcticus, L. othus, and L. timidus (Mammalia: Lagomorpha). Mol Ecol. 2005;14(10):3005–16. https://doi.org/10.1111/j.1365- 294X.2005.02625.x. 30. Alves PC, Melo-Ferreira J, Freitas H, Boursot P. The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus. Philos Trans R Soc Lond Ser B Biol Sci. 2008;363(1505):2831–9. 31. Lopes AM, Blanco-Aguiar J, Martin-Alonso A, Leitao M, Foronda P, Mendes M, Goncalves D, Abrantes J, Esteves PJ. Full genome sequences are key to disclose RHDV2 emergence in the Macaronesian islands. Virus Genes. 2018; 54(1):1–4. https://doi.org/10.1007/s11262-017-1523-2. 32. Gavier-Widén D. Morphological and immunohistochemical characterization of the hepatic lesions associated with European brown hare syndrome. Vet Pathol. 1994;31(3):327–34. 33. Marcato PS, Benazzi C, Vecchi G, Galeotti M, Della Salda L, Sarli G, Lucidi P. Clinical and pathological features of viral haemorrhagic disease of rabbits and the European brown hare syndrome. Rev Sci Tech. 1991;10(2):371–92. 34. Fuchs A, Weissenbock H. Comparative histopathological study of rabbit haemorrhagic disease (RHD) and European brown hare syndrome (EBHS). J Comp Pathol. 1992;107(1):103–13.